Epitope Tagging by Prime Editing in Zebrafish

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Project Summary Recently published search-and-replace genome editing tools utilize the Cas9 nickase to make a single strand break at the target site, followed by introduction of the desired edit by reverse transcriptase using the 3' end of a specifically modified guide RNA (pegRNA) as the template. These tools, named Prime Editors, appear far superior to current state-of-the-art methodologies such as homology directed repair using oligonucleotide templates. It is important to test if this superior performance will reproduce in other model systems including the zebrafish. We propose to use Prime Editors to carry out epitope tagging of several zebrafish genes which play important roles in a variety of biological processes from early embryo patterning to axon guidance and regeneration. Over the course of this work we will determine which Prime Editor has optimal activity in zebrafish, test several important variables of pegRNA design such as the length of the Primer Binding Site, and produce transgenic lines maternally expressing Prime Editor(s), thus facilitating broad application of this method by the research community.